Use of ULIP proteins in the diagnosis and therapy of cancers and paraneoplastic neurological syndromes

ABSTRACT

The invention relates to the use of proteins designated ULIP/POP in the diagnosis and therapy of cancers and paraneoplastic neurological syndromes.

This application is a continuation of U.S. application Ser. No.09/367,496, having a filing date of Nov. 24, 1999, which is a 371 ofPCT/FR98/00328, filed Feb. 19, 1998.

The invention relates to the use of proteins designated ULIP/POP in thediagnosis and therapy of cancers and paraneoplastic neurologicalsyndromes.

Paraneoplastic neurological syndromes (PNS) occur in the instance of acancer, often before its discovery, and are not connected either to thetumour proliferation itself (direct invasion, metastases) or to thetherapy. Their frequency is globally estimated at approximately 1% ofcancers. Several clinical pictures have been individualized for a longtime (encephalomyelitis, Denny-Brown's sensitive neuropathy, cerebellaratrophy, limbic encephalitis, opsoclonus, . . . ) corresponding in factto the either elective or preferential attack of certain groups ofneurons. The frequency of inflammatory cells in the neighbourhood of thelesions for numerous years brought to mind the possibility of anauto-immune or viral process. The more recent demonstration ofauto-antibodies in the serum and the cerebrospinal fluid (CSF) ofpatients suffering from PNS, specific to the type of tumour and the typeof neurons which degenerate, has revived the hypothesis of participationof auto-immunity in the genesis of this pathology (Graus et al., 1985;Greenlee et al., 1983).

Apart from the presence of a high titre of these antibodies in the bloodand the CSF of patients, there are several arguments suggesting that PNSdepend on auto-immune mechanisms. Thus the antigens recognized in thecentral nervous system are also present in the tumours of patients(Anderson et al., 1987). At the level of the tumour tissue, antibodiesspecifically directed against these antigens as well as B and Tlymphocytes are found (Hetzel et al., 1990).

These data suggest that the auto-immune process could be triggered bythe expression of tumour antigens. A cross-immunity process couldprovoke the lesions of the central nervous system. Other argumentsadditionally indicate that the cerebral lesions result from theauto-immune response. Thus, in the brain of the patients, the titre ofspecific antibodies is higher than that of the serum and the CSF (Dalmauet al., 1991). In addition, in the case of encephalomyelitis associatedwith anti-Hu antibodies, there is an intense lymphocytic reaction, madeup of B and T cells, situated in proximity to neurons in the process ofdestruction (Dalmau et al., 1991; Graus et al., 1990).

Several types of auto-antibodies allowing precise syndromic groupings asa function of immunological, neurological and carcinogenic criteria havebeen described.

Thus, anti-Yo antibodies are found in the serum and the CSF of womenhaving paraneoplastic cerebellar atrophy and a gynaecological cancer(ovary, breast or uterus) (Greenlee et al., 1983; Jaeckle et al., 1985).

These antibodies recognize two cytoplasmic proteins of 34 and 62 kDaspecific to Purkinje cells of the cerebellum.

The anti-Ri antibodies are found in the serum and the CSF of patients(principally of women) having opso-myoclonus, cerebellar syndrome andbreast cancer. These antibodies recognize two proteins of 50 and 80 kDaspecific to neurons of the central nervous system (Luque et al., 1991).

Anti-Hu antibodies are most frequently found in the course of PNS. Theyare found in the serum and the CSF of patients having Denny-Brown'ssyndrome or encephalomyeloneuritis and small-cell lung cancer (Graus etal., 1985; Dalmau et al., 1992). These auto-antibodies recognize severalproteins of 37 to 45 kDa expressed specifically by all the neurons ofthe nervous system.

Another type of auto-antibody has recently been identified in patientshaving PNS: anti-CV2 antibodies (Antoine et al., 1993; Honnorat et al.,1996). The latter are atypical, in the sense that the antigenic targetrecognized in adulthood is essentially non-neuronal, although thepost-mortem analysis of the brain of four patients allows neuronal loss,gliosis and an inflammatory process characteristic of PNS to beobjectivized.

The originality of the discovery of these auto-antibodies resides, onthe one hand, in their demonstration. The latter escaped all the usualinvestigations which consisted in revealing the antigens recognized byimmunohistochemistry on post-mortem brain. The antigen recognized isindeed soluble and disappears from post-mortem brain under the majorityof fixation conditions. Only fixation of human post-mortem tissue byimmersion in paraformaldehyde or in situ by perfusion ofparaformaldehyde in animals has allowed the presence of these antibodiesin the CSF or the serum of patients suffering from PNS to be revealed(Antoine et al., 1993; Honnorat et al., 1996).

The anti-CV2 auto-antibodies present in the sera of patients sufferingfrom paraneoplastic neurological syndrome (PNS) have been defined bytheir capacity to recognize, by indirect immunohistochemistry, acytoplasmic antigen expressed specifically, in adult rat brain, by asubpopulation of oligodendrocytes of the brain stem, the medulla and thecerebellum.

The originality of these auto-antibodies resides, on the other hand, intheir diagnostic interest. Their presence in the serum or the CSF ofpatients is of diagnostic value because it allows the paraneoplasticorigin of a neurological syndrome to be specified. The discovery ofthese antibodies, when it precedes that of cancer, directs the search tothat and allows its discovery. Such was the case for six patients out of19 having anti-CV2 antibodies. The clinical disorders were differentaccording to the patients, certain of them having a picture of limbicencephalitis, others encephalomyeloneuritis and others Lambert-Eatonsyndrome. Nevertheless, in more than 60% of the cases, the cerebellarsyndrome was predominant. The most frequently associated tumour wassmall-cell lung cancer (60% of the cases).

Experiments on the brains of newborn rats showed that these anti-CV2antibodies reacted with a protein of 66 kDa (Honnorat et al., 1996).

In the adult brain, this antigen is situated in a subpopulation ofoligodendrocytes or in cells which retain differentiation capacities inthe adult brain (olfactory bulb, dentate gyrus). The recognized antigencould play a role in neuronal survival, via Neuron/Oligodendrocyteinteractions, as the loss of neurons observed in the post-mortem brainof patients suffering from PNS suggests.

Its very limited expression in adulthood contrasts with a very strongand transitory expression in the central and peripheral nervous systemin development, suggesting the probable role of this antigen in thedevelopment of the nervous system.

The Applicant has characterized the target antigen of anti-CV2antibodies, which corresponds to a protein designated below by “POP-66”for “paraneoplastic oligodendrocyte protein 66 kDa”.

Surprisingly, it has been discovered that the POP-66 protein belongs tothe so-called ULIP family of proteins (for Unc-33-like phosphoprotein),involved in the control of neuronal development and axonal transport (T.Byk et al., 1996), and also studied in the form of CRMP proteins(Goshima et al., 1995, Wang et al., 1996), TOAD-64 (Minturn et al.,1995) and DRPs (Hamajima et al., 1996). More precisely, POP-66 has beenidentified as in fact being the human form of ULIP-4.

All of the data described below emphasize the complexity of this familyof proteins, the existence of a very wide expression spectrum of membersof this family in the brain in the course of ontogenesis, but a verylimited spectrum in adults, as well as the specificity of the anti-CV2antibodies for a member of this ULIP protein family, which is in factPOP-66.

Thus, the Applicant has shown that the protein recognized by theanti-CV2 antibodies of patients suffering from PNS is POP-66/ULIP-4 andhas established the involvement of the ULIP proteins in paraneoplasticneurological syndromes and associated cancers. In addition to their rolein cancers associated with PNS, the Applicant has likewise discoveredthat the proteins of the ULIP family could play a role in any other formof cancer not associated with PNS. More particularly, the ULIP proteinscould especially be involved in cancers of tissues having a commonembryonic origin with the central nervous system.

The present invention therefore relates to a purified ULIP polypeptide,derivative or polypeptide fragment of the said purified polypeptide,comprising an amino acid sequence selected from SEQ ID No. 2, No. 4, No.6 and No. 8.

Preferentially, the present invention relates to a purified polypeptide,derivative, or biologically active polypeptide fragment of the saidpurified polypeptide, comprising the amino acid sequence SEQ ID No. 8,the said polypeptide being designated by “POP-66/ULIP-4”.

A fragment of the polypeptide of sequence SEQ ID No. 8 of interest is,in particular, the antigenic fragment PARASCPGKIS (amino acids No. 517to No. 527).

In the description of the invention, the following definitions are used:

-   -   derivative: any variant polypeptide of the polypeptide of        sequence SEQ No. 2, No. 4, No. 6 or No. 8 or any other molecule        resulting from a modification of genetic and/or chemical nature        of the sequence SEQ ID No. 2, No. 4, No. 6 or No. 8, that is to        say obtained by mutation, deletion, addition, substitution        and/or chemical modification of a single or of a limited number        of amino acids, as well as any isoform sequence, that is to say        a sequence identical to the sequence SEQ ID No. 2, No. 4, No. 6        or No. 8, to one of its fragments or modified sequences,        containing one or more amino acids in the D enantiomer form, the        said modified or isoform variant sequences having conserved at        least one of the properties making them biologically active.    -   biologically active: having properties of induction and/or        control of neuronal development and/or antigenic properties.

The invention likewise relates to an isolated nucleic acid sequenceselected from SEQ ID No. 1, No. 3, No. 5 and No. 7 or a sequence derivedfrom the sequences SEQ ID No. 1, No. 3, No. 5 and No. 7 on account ofthe degeneracy of the genetic code, or on account of mutation, ofdeletion or of insertion of at least one nucleotide, the said derivedsequences having a biological activity virtually identical to that ofthe peptide encoded by the sequences SEQ ID No. 1, No. 3, No. 5 and No.7.

The various nucleotide sequences of the invention can be of artificialor non-artificial origin. They can be DNA or RNA sequences, obtained byscreening of banks of sequences by means of probes elaborated on thebasis of sequences selected from SEQ ID No. 2, No. 4, No. 6 and No. 8.Such banks can be prepared by conventional techniques of molecularbiology known to the person skilled in the art.

The nucleotide sequences according to the invention can likewise beprepared by chemical synthesis, or alternatively by mixed methodsincluding the chemical or enzymatic modification of sequences obtainedby screening of banks.

These nucleotide sequences allow the production of nucleotide probescapable of hybridizing strongly and specifically with a nucleic acidsequence of a genomic DNA or of a messenger RNA coding for a peptideaccording to the invention or a biologically active fragment of this.The appropriate hybridization conditions correspond to the conditions oftemperature and of ionic strength usually used by the person skilled inthe art (Sambrook et al., 1989), preferably to temperature conditions ofbetween (T_(m) minus 5° C.) and T_(m) minus 30° C.) and more preferablyto temperature conditions of between (T_(m) minus 5° C.) and (T_(m)minus 10° C.) (great stringency), T_(m) being the theoretical meltingpoint, defined as being the temperature at which 50% of the pairedstrands separate. Such probes are likewise part of the invention. Theycan be used as a diagnostic tool in vitro for the detection, byhybridization experiments, of specific transcripts of polypeptides ofthe invention in biological samples or for the demonstration of aberrantsyntheses or genetic anomalies resulting from polymorphism, mutations orbad splicing.

The probes of the invention contain at least 10 nucleotides, and at mostcontain the whole of a nucleotide sequence selected from SEQ ID No. 1,No. 3, No. 5 and No. 7 or of their complementary strand.

The in vitro diagnostic methods in which these nucleotide probes areemployed for the detection of aberrant syntheses or genetic anomalies,such as the loss of heterozygosity and genetic rearrangement, at thelevel of nucleic sequences coding for a ULIP polypeptide according tothe invention or a biologically active fragment are included in thepresent invention. Such a method type comprises:

-   -   the contacting of a nucleotide probe of the invention with a        biological sample under conditions allowing the formation of a        hybridization complex between the said probe and the        abovementioned nucleotide sequence, optimally after a previous        amplification step of the abovementioned nucleotide sequence;    -   the detection of the hybridization complex optimally formed;    -   optimally the sequencing of the nucleotide sequence forming the        hybridization complex with the probe of the invention.

The cDNA probes of the invention can additionally be advantageously usedfor the detection of chromosomal anomalies.

The nucleotide sequences according to the invention are likewise usefulfor the production and use of sense and/or antisense oligonucleotideprimers for sequencing reactions or specific amplification reactionsaccording to the so-called PCR technique (polymerization chain reaction)or any other variant of this.

The nucleotide sequences according to the invention additionally haveuses in the therapeutic field, for the production of antisense sequencescapable of hybridizing specifically with a nucleic acid sequence,including a messenger RNA, which can be used in gene therapy. Theinvention thus relates to antisense sequences capable of inhibiting, atleast partially, the production of a polypeptide according to theinvention, such as defined above.

They are more particularly useful in the treatment of disorders of thecentral and peripheral nervous system and of vision, especially in thetreatment of paraneoplastic neurological syndromes, as well as inanti-cancer treatment, especially of tumours associated withparaneoplastic neurological syndromes.

The nucleotide sequences according to the invention can additionally beused for the production of recombinant ULIP proteins according to theinvention.

These proteins can be produced from nucleotide sequences defined above,according to techniques of production of recombinant products known tothe person skilled in the art. In this case, the nucleotide sequenceused is placed under the control of signals allowing its expression in acell host.

An efficacious system of production of a recombinant proteinnecessitates having a vector, for example of plasmid or viral origin,and a compatible host cell.

The cell host can be selected from prokaryotic systems, such asbacteria, or eukaryotic systems, such as, for example, yeasts, insectcells, CHO (Chinese hamster ovary) cells or any other systemadvantageously available. A preferred cell host for the expression ofproteins of the invention is formed by the bacterium E. coli.

The vector must contain a promoter, translation initiation andtermination signals, as well as the appropriate regions of transcriptionregulation. It must be able to be maintained stably in the cell and canpossibly possess special signals specifying the secretion of thetranslated protein.

These different control signals are selected as a function of the cellhost used. To this end, the nucleotide sequences according to theinvention can be inserted in autonomous replication vectors within theselected host, or integrative vectors of the selected host. Such vectorswill be prepared according to methods currently used by the personskilled in the art, and the resulting clones can be introduced into anappropriate host by standard methods, such as, for example,electroporation.

The invention is additionally directed at the host cells transfected bythese above vectors. These cells can be obtained by the introductioninto host cells of a nucleotide sequence inserted into a vector such asdefined above, then the culturing of the said cells under conditionsallowing the replication and/or expression of the transfected nucleotidesequence.

These cells can be used in a method of production of a recombinantpolypeptide according to the invention or any fragment or biologicallyactive derivative of this.

The method of production of a polypeptide of the invention inrecombinant form is itself included in the present invention, and ischaracterized in that the transfected cells are cultured underconditions allowing the expression of a recombinant polypeptideaccording to the invention or of any fragment or biologically activederivative of this, and in that the said recombinant polypeptide isrecovered.

The purification processes used are known to the person skilled in theart. The recombinant polypeptide can be purified from lysates and cellextracts, from the supernatant of the culture medium, by methods usedseparately or in combination, such as fractionation, chromatographicmethods, immunoaffinity techniques with the aid of specific mono- orpolyclonal antibodies, etc.

One variant consists in producing a recombinant polypeptide fused to a“carrier” protein (chimeric protein). The advantage of this system isthat it allows a stabilization and a decrease in the proteolysis of therecombinant product, an increase in the solubility in the course of thein vitro renaturation and/or a simplification of the purification whenthe fusion component has an affinity for a specific ligand.

The exploitation of ULIP proteins, and in particular POP-66/ULIP-4, aswell as antibodies directed against these proteins, is promising invarious fields.

Thus, the detection of the anti-CV2 auto-antibody by immunofluorescenceon fixed animal brain is currently used as a diagnostic test.

The production of POP-66/ULIP-4 recombinant protein according to theinvention allows the production of a rapid and reliable test (of Elisaor Western Blot type) for detecting anti-CV2 antibodies.

Such tests already exist for anti-Hu, anti-Yo and anti-Ri antibodies.The test for detecting anti-CV2 in the serum of patients could beprescribed in the case of suspicion of paraneoplastic neurologicalsyndrome and consequently could include anti-CV2 antibodies at the sametitre as the other antibodies identified in the PNS such as mentionedabove.

The invention is therefore likewise directed at a method for thediagnosis of paraneoplastic neurological syndromes and/or for the earlydiagnosis of the formation of tumours of cancerous origin, characterizedin that auto-antibodies directed against a POP-66/ULIP-4 protein aredemonstrated in a blood sample taken from an individual by

-   -   the contacting of a blood sample taken from an individual with a        purified polypeptide (POP-66), derivative or biologically active        polypeptide fragment of POP-66/ULIP-4 optionally attached to a        support under conditions allowing the formation of specific        immunological complexes between the said polypeptide and the        auto-antibodies optionally present in the serum sample, and    -   the detection of the specific immunological complexes optionally        formed.

The invention likewise relates to a kit for the diagnosis ofparaneoplastic neurological syndromes and for the early diagnosis of theformation of tumours from a biological sample, comprising:

-   -   at least one purified POP-66/ULIP-4 polypeptide, derivative or        biologically active polypeptide fragment of POP-66/ULIP-4,        optionally attached to a support,    -   means of visualization of the formation of specific        antigen/antibody complexes between an anti-POP-66 auto-antibody        and the said purified POP-66 polypeptide, derivative or        polypeptide fragment and/or means of quantification of these        complexes.

The invention likewise relates to mono- or polyclonal antibodies ortheir fragments, chimeric or immunoconjugated antibodies obtained from apurified ULIP polypeptide comprising an amino acid sequence selectedfrom SEQ ID No. 2, No. 4, No. 6 and No. 8, derivative or biologicallyactive polypeptide fragment of ULIP and their use for the purificationor the detection of a ULIP protein in a biological sample.

Polyclonal antibodies can be obtained from the serum of an animalimmunized against the protein, produced, for example, by geneticrecombination according to the method described above, according to theusual working methods.

The monoclonal antibodies can be obtained according to the conventionalmethod of hybridoma culture described by Köhler and Milstein.

The antibodies can be chimeric antibodies, humanized antibodies, Fab andF(ab′)₂ fragments. They can likewise be present in the form ofimmunoconjugates or labeled antibodies.

The invention likewise relates to the use of antibodies directed againsta protein of the ULIP family for the demonstration of a ULIP protein inneoplasms, and paraneoplastic neurological syndromes for diagnosticpurposes.

Preferentially, the invention relates to the use of monoclonalantibodies obtained from polyclonal anti-CV2 serum of patients byimmortalization of lymphocytes, according to the usual techniques knownto the person skilled in the art.

Thus, the antibodies directed against a protein of the ULIP family areuseful for detecting abnormal expression of ULIP protein in patientshaving neurological syndromes, in whom cancer has not been diagnosed bythe conventional methods. This abnormal expression of ULIP protein willbe able to be correlated with the existence of a cancer which had notbeen spotted. Thus, the antibodies directed against a ULIP protein,especially against POP-66/ULIP-4, are useful for the early diagnosis ofcancer.

The invention likewise relates to a method of determination of anallelic variability, a mutation, a deletion, an insertion, a loss ofheterozygosity or a genetic anomaly of the POP-66/ULIP-4 gene, situatedon chromosome 10 in the 26q region and which can be involved inpathologies, characterized in that it employs at least one nucleotidesequence SEQ ID No. 7. Amongst the methods of determination of anallelic variability, a mutation, a deletion, an insertion, a loss ofheterozygosity or a genetic anomaly of the POP-66/ULIP-4 gene, a methodcomprising at least one PCR amplification step of the nucleic sequenceof POP-66/ULIP-4 capable of having a polymorphism, a mutation, adeletion or an insertion with the aid of pairs of primers of nucleotidesequences, a step in the course of which amplified products are treatedwith the aid of appropriate restriction enzymes and a step in the courseof which at least one of the products of the enzymatic reaction isdetected or determined is preferred.

Advantageously, it is possible to search for the mutations associatedwith the said chromosome 10 in relation to cancer, especially peripheralcancerous tumours and primitive cerebral tumours of glial origin, forexample.

The invention likewise relates to a pharmaceutical compositioncomprising at least one purified protein of the ULIP family, polypeptidefragment or biologically active derivative of this, a nucleotidesequence or nucleotide sequence fragment coding for the said protein, anantisense sequence capable of hybridizing specifically with a nucleotidesequence coding for the said protein, or an antibody directed againstthe said protein, combined with a pharmaceutically acceptable vehicle.

The invention preferentially comprises pharmaceutical compositionscomprising as active principle a purified POP-66 polypeptide, derivativeor polypeptide fragment of POP-66, preferentially in soluble form,combined with a pharmaceutically acceptable vehicle.

Such compositions offer a new approach to treating disorders of thecentral and peripheral nervous system and of vision, and especiallyparaneoplastic neurological syndromes. In addition, they are useful fortreating neurological disorders connected with a neuronal loss and/or anunderexpression of ULIP proteins in the nervous system.

Thus, POP-66/ULIP-4 is also of interest in neurodegenerative pathologiessuch as multisystemic atrophies which are conditions similar to those ofPNS and for which an anomaly of an oligodendrocytic subpopulation hasbeen detected (Papp et al., 1992).

The compositions according to the invention are additionally useful inanticancer therapy.

The antibodies directed against one or more ULIP proteins can becombined with antineoplastic agents, thus allowing the targeting ofmedicaments towards the tumour cells.

They can additionally be combined with a hydrophilic chemical groupchosen in such a way so as to cross or not to cross the blood-brainbarrier, according to the type of tumour.

The ULIP proteins and in particular POP-66 as well as the nucleotidesequences coding for the said proteins and the antisense sequences oroligonucleotides can be useful in the therapy of any type of cancer inwhich a gene coding for a ULIP protein is involved. Amongst examples ofcancers, it is possible to mention peripheral tumours, such assmall-cell lung cancer, thymoma, cancer of the breast and of the ovary,as well as cerebral tumours, preferably primitive cerebral tumours ofglial origin. The expression of POP-66 in the non-proliferative cells ofnormal brain, its absence in normal tissues such as lung or thymus, forexample, its differential reexpression during tumorigenesis of thesetissues and the modulation of its expression in a tumour line in thecourse of differentiation suggest in this respect that POP-66 could be atumour suppressor gene.

Preferentially, the pharmaceutical compositions according to theinvention can be administered by the systemic route, preferably by theintravenous route, by the intramuscular route, intradermally or by theoral route.

Their modes of administration, dosages and optimal pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a therapeutic treatment adapted to a patient,such as, for example, the age or the body weight of the patient, theseriousness of his/her general condition, the tolerance to the treatmentand the secondary effects noted, etc.

The invention likewise comprises the use of a purified protein of theULIP family, polypeptide fragment or biologically active derivative ofthis, a nucleotide sequence or nucleotide sequence fragment coding forthe said protein, an antisense sequence capable of hybridizingspecifically with a nucleotide sequence coding for the said protein, oran antibody directed against the said protein, combined with apharmaceutically acceptable vehicle, for the production of a medicamentintended for treating neurodegenerative illnesses and neoplasms.

The invention finally relates to a method of treatment ofneurodegenerative illnesses and neoplasms, comprising the administrationto a subject requiring such a treatment of a therapeutically efficaciousquantity of a purified protein of the ULIP family, polypeptide fragmentor biologically active derivative of this, a nucleotide sequence ornucleotide sequence fragment coding for the said protein, an antisensesequence capable of hybridizing specifically with a nucleotide sequencecoding for the said protein, or an antibody directed against the saidprotein, combined with a pharmaceutically acceptable vehicle.

The examples and the figures whose legends are presented below are givenby way of illustration.

LEGEND TO THE FIGURES

FIG. 1 represents a two-dimensional electrophoresis profile obtainedfrom brain protein extracts of newborn rats enriched in POP-66.

A: silver staining of all of the proteins.

B: immunoblot with the anti-CV2 serum of patients.

The arrows indicate the spots corresponding to POP-66, revealed withanti-CV2 antibodies.

FIG. 2 represents a two-dimensional electrophoresis profile obtainedfrom protein extracts of brains of newborn rats.

Immunoblot with A-antipeptide antibody 3 and B-anti-CV2 antibody.

FIG. 3 represents a one-dimensional electrophoresis obtained fromprotein extracts of brains of newborn rats.

Immunoblot with a: preimmune serum for peptide 3

Immunoblot with b: anti-peptide serum 3

Immunoblot with c: anti-peptide serum 4

Immunoblot with d: preimmune serum for peptide 4.

FIG. 4 represents an immunohistochemical labelling of sections of brainsof adult rats with

A: anti-CV2 serum of a patient suffering from PNS

B: rabbit serum with anti-peptide 3 antibodies

C: rabbit serum with anti-peptide 4 antibodies.

FIG. 5 represents a histological labelling of sections of young ratcerebellum 8 days post-natally.

A: Staining with toluidine blue; ge=external granular layer; m=molecularlayer (×400).

B: Immunolabelling after incorporation of BrdU (bromodeoxyuridine). Thecells which have incorporated BrdU are virtually all situated in themost external zone of the external granular layer (ge). Some positivecells are situated in the internal granular layer (×400).

C: Indirect immunoperoxidase with a patient serum containing an anti-CV2antibody (×400). The immunoreactivity is concentrated in the internalpart of the external granular layer (future molecular layer (m)). Somecells are immunoreactive in the internal granular layer. The Purkinjecells (p) are negative as well as the cells of the external part of theexternal granular layer (ge).

D: Indirect immunoperoxidase with a patient serum containing an anti-CV2antibody (×1000). Above all, the immunolabelling is concentrated in theinternal part of the external granular layer (future molecular layer(m)). A reactive cell is noted in the internal granular layer (gi)(arrow).

FIG. 6 represents immunohistochemical labelling of sections ofpost-mortem hippocampus (HPS staining).

A: brain of control patient,

B: brain of patient having limbic encephalitis, and circulating anti-CV2antibody. It is possible to note the disappearance of the granularcells.

FIG. 7 represents a two-dimensional electrophoresis profile with thecontrol ULIP-2 protein (A) and the ULIP-4 protein (B).

FIG. 7C represents the migration profile model of the proteins ULIP-1,2, 3 and 4 as a reference.

The proteins are revealed:

a) by autoradiography to locate the proteins translated in vitro(translation);

b) by immunoblotting with the anti-CV2 serum.

FIG. 8 represents a migration profile of the mRNA of C-22/ULIP-3 (8A)and TOAD-64/ULIP-2 (8B) amplified by RT-PCR expressed in different celltypes:

lanes 1-3: small-cell lung tumour

lane 2: small-cell lung tumour with anti-CV2 serum

lane 4: control cDNA.

lane 5: medulloblastoma treated by HTLV1 infection

lanes 6-7: medulloblastoma

lane 8: C6 line of glial cells in mice

lane 9: control

lane 10: nothing

lane 11: kb scale.

The black arrows correspond to POP-66; the white arrows correspond tothe molecular weight standard.

FIG. 9 represents the nucleotide sequence of ULIP-2 in mice (SEQ ID No.1), as well as the inferred amino acid sequence (SEQ ID No. 2).

FIG. 10 represents the nucleotide sequence of ULIP-3 in mice (SEQ ID No.3), as well as the inferred amino acid sequence (SEQ ID No. 4).

FIG. 11 represents the nucleotide sequence of ULIP-4 in mice (SEQ ID No.5), as well as the inferred amino acid sequence (SEQ ID No. 6).

FIG. 12 represents the nucleotide sequence of ULIP-4 in man (SEQ ID No.7), as well as the inferred amino acid sequence (SEQ ID No. 8).

An erroneous stop codon in the human ULIP-4 sequence (asterisk) arisesfrom a fault of the reverse transcriptase in the production of the bank.By comparing with ULIP-4 of rats and of mice, it is almost certain thatthe TAG sequence coding for a stop is in fact an AAG codon, coding for alysine as in rats and mice. In addition, the region around this aminoacid is entirely conserved in the three species.

The amino acid sequence has been completed in SEQ ID No. 8 by 15C-terminal amino acids (No. 554 to No. 568). This C-terminal regionwhich is missing in FIG. 12 is very well conserved between rat and miceULIP-4 as well as between the different ULIPs.

EXAMPLE 1 Purification of POP-66 and Sequencing

The purification of POP-66 is carried out according to the material andthe methods described in the article of Honnorat et al., 1996,incorporated by reference, starting from serum of patients sufferingfrom PNS.

To identify the protein POP-66, a purification strategy was chosen whichallows a partial sequencing to be obtained. The screening of anexpression bank of brain cDNA or the immunoaffinity purification of theprotein were excluded because of the limited quantities of sera linkedto the death of the patients. It was possible to develop a method ofbiochemical purification starting from brains of newborn rats on accountof the anti-CV2 human sera, which allowed each purification step to bemonitored.

The tissues, preserved at −70° C. before use, were treated with asolution containing 0.2 M DTT (dithiothreitol) (Sigma) 2% Ampholine 3-10(Pharmacia), 2% Triton X-100 (Merck) and placed at 2-4° C. Immediatelybefore use, solid urea (Pharmacia) was added to obtain an 8M solution.

The POP-66 protein is soluble, at least in part, and precipitatesentirely at a concentration of 40% ammonium sulphate.

Centrifugation at 100,000 (times) g and ammonium sulphate precipitation(eliminating the proteins precipitating below 20% and above 40% ammoniumsulphate) allows protein extracts enriched in POP-66 to be obtained. Theproteins of this extract are then separated, after dialysis, byisofocusing on agarose gel (Peltre et al., 1982).

After transfer to a membrane, the anti-CV2 antibodies recognize severalbands of isoelectric points of between 5.85 and 6.55. All of these bandscorrespond to the POP-66 protein recognized by the anti-CV2 antibodies.This spectrum suggests the possibility of transcriptional modifications(phosphorylations and/or glycosylations) of the protein.

The zone of proteins of pI between 5.85 and 6.55 from the agarose gel isused for a new electrophoretic migration in denaturating medium onpolyacrylamide gel previously equilibrated with an equilibrationsolution (0.05 mol/1 Tris/HCl, pH 6.8, 6M urea, 30% glycerol, 1%weight/volume SDS for 2×10 minutes) to which is added DTT (0.25%weight/volume) and bromophenol blue.

Two methods of detection are used:

-   -   silver staining. Immediately after the end of the migration, the        gel is immersed in a fixing solution (40% ethanol, 10% acetic        acid) for 30 minutes; it is then placed in an incubation        solution (30% ethanol, 7% weight/volume of sodium acetate, 0.1%        glutaraldehyde, 0.2% weight/volume of sodium thiosulphate) for        30 minutes or one night. After washing, the gel is placed in a        silver solution (0.1% weight/volume of silver        nitrate+formaldehyde) and developed (2.5% weight/volume of        sodium carbonate +formaldehyde). The reaction is stopped with        Na₂ EDTA (1.5% weight/volume). The gels are preserved in a        glycerol solution.    -   transfer to a PVDF membrane (Immobilon-P®, Millipore). The        separated proteins are transferred to a PVDF membrane using a        100 mM CAPS buffer (Sigma) of pH 11. The transfers are incubated        for one hour in TBS buffer (Tris buffer saline) with 5% of        casein (milk) and 18 hours in TBS buffer (+1% of casein)        containing antibody ( 1/500 anti-CV2 serum). After washing with        TBS-casein (15 minutes), visualization is carried out by        incubating the transfers for 1 and a half hours with        biotinylated anti-IgG antibodies ( 1/1000) and for 1 and a half        hours with the streptavidin-peroxidase complex ( 1/2000). The        transfers are then visualized with DAB (0.06% weight/volume        diaminobenzidine in 0.05 M Tris) and with H₂O₂ (0.02 μg/ml).

A single band corresponding to a protein of 66 kDa is visible. This isspecifically labeled with anti-CV2 antibodies (FIG. 1). An N-terminalsequencing of this protein was then carried out, after trypsicdigestion.

Seven peptides, having the following sequences, were obtained: 1X-Met-Tyr-Asp-Gly-Pro 2 X-Phe-Asn-Leu-Tyr-Pro-Arg 3X-Val-Leu-Glu-Asp-Gly-Thr-Leu-His-Val-Thr-Glu-Gly 4X-Ile-Gly-X-X-Ala-Gln-Val-(His?)-Ala-Glu-Asn-Gly-X-Ile-Ile-Ala-Glu-Glu-Gln 5 X-X-Glu-Asn-Gln-Phe-Val-Ala-Val-Thr 6X-Val-Asn-Asp-(Asp?)-Gln-Ser-Phe-Tyr-Ala-Asp-Ile-Tyr-Met-Glu-(Asp?)-(Gly?)-Leu-Ile 7 X-X-X-Phe-Val-Thr-X-Pro-X-Leu-X-Pro

-   X: corresponds to a non-determined amino acid,-   (?): corresponds to a probable but uncertain amino acid.

According to the analysis of databanks available in 1994, no knownprotein corresponded to these sequences.

EXAMPLE 2 Cloning of the cDNA of POP-66 or of Related Proteins

The cloning of the cDNA of the POP-66 protein or of related proteins wascarried out by using degenerate oligonucleotide probes obtained fromfragments of two peptides:

-   Ile-Ile-Ala-Glu-Glu-Gln-   Tyr-Ala-Asp-Ile-Tyr-Met-Glu-(Asp ?)

Four sets of degenerate oligonucleotide primers (sense/antisense) aretherefore determined

-   (AT(C/T)ATTGC(T/A)GA(A/G)CA;TG(C/T)TC(T/C)AC(T/A)GCAT(A/G)AT;-   TATGC(A/T)GA(C/T)AT(C/T)ATGGA; TCCAT(G/A)TA(G/A)CT(T/A)GCATA, and    used for a PCR amplification.

The matrix is prepared in the form of double-stranded cDNA (Promega kit)from poly(A⁺)RNA extracted from the brain of rats 10 days old(Zivic-Miller, USA) using the Fast Track mRNA isolation kit(Invitrogen).

The conditions of amplification by PCR are as follows: 35 cycles at 94°C., 1 minute for denaturation, 55° C., 1 minute for hybridization and72° C., two minutes, for extension.

The PCR products are analysed by 1% agarose gel electrophoresis,electroeluted, cloned in a TA cloning vector (Invitrogen) and sequencedusing the primer sites of the T7 and SP6 promoters.

The sequence of amino acids inferred from the MFB-17 clone agrees withthe sequences of the two original peptides of POP-66 determined by theanalysis of the amino acid sequence.

A comparative analysis of the nucleic acid sequences using the Genbankand EMBL databases reveals that MFB-17 is a partial cDNA with anucleotide sequence identical to that of a segment of TOAD-64, a ratneuronal protein (Minturn et al., 1995).

The amino acid sequence inferred in the cDNA of TOAD-64 agrees with thesequences of the seven peptides determined by partial sequence analysisof the protein recognized by the anti-CV2 antibodies after purificationby electrophoresis.

The molecular weight, the isoelectric point, the immunohistochemicalprofile and the regulation of TOAD-64 are similar to those of the POP-66antigen.

Since the MFB-17 clone did not have the complete coding region, it wasnecessary to produce an intact recombinant protein to continue theresearch concerning the CV2 protein.

To obtain a complete TOAD-64 protein, the ds-cDNA matrix of rat brainswas amplified with two sets of primers situated at the 5′ and 3′extremities of the coding regions (sense: GGCATATGTCTTATCAGGGGAAG;antisense: GCGAATTCTTAGCCCAGGCTGATG).

This approach allowed two different clones to be produced, onecorresponding to the TOAD-64 sequence and the other to a clonedesignated by C-22.

EXAMPLE 3 Comparison of the Amino Acid Sequence Inferred From C-22 withthe ULIP Proteins

The amino acid sequence inferred from the open reading frame indicatesthat this C-22 clone belongs to the superfamily of ULIP genesrepresented by several genes of EST sequences.

The amino acid sequence inferred from C-22 has a homology of 30% withthe amino acid sequence of the unc-33 protein of Caenorhabditis elegans.

Recently, four different homologous genes in the unc-33 protein havebeen described in mammals and the chicken.

An analysis of the sequences by the Genbank databases and protein bankshas allowed a classification of the unc-33-like (ULIP) proteins intofour different subgroups to be proposed (Byk et al. 1996).

However, as the real functions of these proteins are not clearly known,the proposed classification is simply based on the percentage ofidentity of amino acids. ULIP-1 is represented by a mouse “unc-33-like”phosphoprotein which has a homology of 76% with TOAD-64, Crmp-62 andMunc, a mouse sequence recently available from Genbank.

ULIP-2 is composed of TOAD-64, Crmp-62 and Munc which between them havea 97% amino acid identity.

The partial human EST sequences, that is to say hcrmp-1, which have a75% identity with ULIP-1 or ULIP-2, have been found. They belong to athird group called ULIP-3. The last group identified called ULIP-4comprises r-CRMP-3 in the rat and the forms ULIP-4 in the mouse andPOP-66/ULIP-4 in man.

The comparison of the amino acid sequence of the three ULIP genes,namely TOAD-64 in the rat, Crmp-62 in the chicken and ULIP-1 in themouse, with the amino acid sequence inferred from the open reading frameof the present C-22 clone, using the Clustal V alignment softwareprogram reveals that C-22 has an identity of 74% with ULIP-1, 77% withCrmp-62 and 76% with TOAD-64.

The nucleotide sequence C-22 has an identity of 97% with the partialsequence EST, hCrmp-1, and thus defines the third member of the ULIP-3group. The TOAD-64, Crmp-62 and C-22 genes each code for a protein of572 amino acids in length, whereas the amino acid sequence inferred fromULIP-1 gives a protein of 570 amino acids.

The analysis of the amino acid sequence of C-22 does not show any signalsequence or transmembrane domain suggesting that the product(s) of theC-22 gene could be localized in the cytoplasm of the cells.

Several consensus sites of phosphorylation by the kinase C protein (S/TX R/K) appear along the length of the product of the C-22 gene. Theseobservations suggest that C-22 is a phosphoprotein and that the slightdifferences in the phosphorylation could dictate the activity or therole of different members of this family of proteins throughout the cellcycle. TABLE I Summary of proteins having a homology with the ULIPs.Family Species EMBL No. Nematode Unc-33 Nematode Z14146Dihydropyrimidinase Hu DHPase human D78011 Ra DHPase rat D63704 ULIP-1group Ulip mouse X87817 Hu DRP3 human D78014 r-CRMP-1 rat U52102 Hu-Uliphuman Y07818 ULIP-2 group ULIP-2 mouse SEQ ID No. 2 Toad-64 rat Z46882CRMP-62 chicken U17277 Munc mouse X87242 HCRMP-2 human U17279 Hu DRP-2human D78013 r-CRMP-4 rat U52104 ULIP-3 group ULIP-3 mouse SEQ ID No. 4HCRMP-1 human U17278 rCRMP-1 rat U52102 C-22 rat U52095 Hu DRP-1 humanD78012 ULIP-4 group ULIP-4 mouse SEQ ID No. 6 POP-66/ULIP-4 man SEQ IDNo. 8 r-CRMP-3 rat U52103

EXAMPLE 4 Regulation of the Expression of the C-22 Gene

The evaluation of alterations in the expression of the C-22 gene couldhave considerable significance for the knowledge of the functionalaspects of the C-22 protein.

Consequently, the Applicant studied the possible regulation of theexpression of the C-22 gene in the course of development. The total RNAis extracted and separated by electrophoresis on 1% agarose gel andtransferred to Nytran membrane (Duchemin et al. 1987). The transfers arehybridized with a C-22 coding sequence labelled with ³²P, a 0.5 mMphosphate buffer and 5% SDS at 65° C. for 16 hours.

At the end of the hybridization, the transfers are washed successivelythree times with 2×SSC, 0.1% SDS at ambient temperature, then 1×SSC,0.1% SDS at 65° C. for 60 minutes, and exposed to X-rays.

Under the conditions used, a single band at 3.8 kb was detectedrepresenting the C-22 mRNA which is also the smallest transcript of theunc-33 family of genes of vertebrates. The size of the transcriptremains the same during the pre- and post-natal periods.

The kinetics of the C-22 gene in the brain of rats in the course ofdevelopment shows that the messenger is detectable in the course of theembryonic period on day E17. The quantity of C-22 transcripts increasesup to day 7 post-natally then decreases rapidly from the second weekafter birth to a virtually undetectable level in the adult.

Around birth, a still unknown regulation signal is probably received,which increases the expression of the C-22 gene, this signal beingtemporarily linked to neuronal differentiation and to axonaldevelopment.

The mRNA of C-22 has not been able to be detected by Northern Blotanalysis in several regions of the brain such as the frontal cortex, themidbrain and the thalamus in adults and rats more than two years old.

In addition, it has not been possible to detect the mRNA of C-22 innon-neuronal tissues, such as the heart, the lung, the liver and thekidney in one-week old rats and adult rats.

The data on the expression profile of the mRNA of C-22 suggests adecisive role of the C-22 protein in the development of the brain.

EXAMPLE 5 Immunoblotting of POP-66

A—Materials and Methods

Transfection of the ULIPs in E. coli

The full-length cDNAs of rat ULIP-2 and ULIP-3 and mouse ULIP-1 andULIP-4 were subcloned directionally in the E. coli pET-21a(+) expressionvector after introduction of a 5′ Nde I site and of a 3′ EcoRI site byPCR, and the four constructs were resequenced. The expression of thetarget gene induced by IPTG was carried out according to the protocol ofthe manufacturer (Novagen).

Production of Anti-ULIP Antibodies

Rabbit antibodies (anti-Pep3) are directed against the peptideITGPEGHVLSRPEEVE (amino acids 217-232 of the sequence SEQ ID No. 8),synthesized in a multiple peptide synthesis apparatus using F-moc (432APeptide Synthesizer SYNERGY, Applied Biosystems). The purity was checkedby analysis of the sequence by HPLC and mass spectrometry. 1 mg of thesynthetic peptide conjugated to limpet haemocyanin, in complete Freund'sadjuvant, was used to immunize rabbits with a booster dose of 0.5 mg ofbound peptide in complete Freund's adjuvant after 4 weeks. The anti-Pep3antibodies recognized the four recombinant ULIP proteins expressed in E.coli.

Labelling with the anti-Pep3 antibodies was removed after preincubationwith peptide 3. Controls with pre-immune rabbit sera were negative.

Anti-peptide 4 antibodies directed against the peptide LEDGTLHVTEGS wereproduced according to the same protocol.

B—Results

Antibodies against four of the sequenced peptides were products. Two ofthe sera turned out to be of particular interest.

One contains antibodies (Ab anti-pep3) which recognize several membersof the ULIP family on two-dimensional electrophoresis of proteinextracts of newborn rat brain (FIG. 2) and on one-dimensionalelectrophoresis (FIG. 3). On Western Blotting, another antibody (Abanti-Pep4) recognizes a single band of 66 kDA capable of correspondingto a single member of the family (FIG. 3), namely ULIP-2.

EXAMPLE 6

Immunohistochemistry

The tissue preparations for immunohistochemistry are obtained fromnewborn rat brains and from post-mortem human brains, fixed at 4° C. in4% paraformaldehyde and 0.2% picric acid diluted in phosphate buffer(0.1 M, pH=7.4), then cryoprotected.

Immunocytochemistry can be carried out by the indirectimmunofluorescence technique. Sections of 12 μm in thickness areprepared in a cryostat and then mounted on gelatin-covered slides,treated with 0.1% Triton X100 for 2 hours in PBS buffer and 1% bovineserum albumin (BSA) and incubated for 12 h with anti-CV2 serum ofpatients in PBS/1% BSA at ambient temperature ( 1/100 dilution of theserum). After several washes with PBS/1% BSA, the sections are incubatedfor 2 h with a rabbit anti-human antiserum conjugated to fluoroscein,diluted to 1% (Dakopatts) in PSB/1% BSA. After washing in PBS, theslides are examined under the microscope. The control sections areincubated either with anti-human IgG antiserum conjugated to fluorosceinalone, or PBS/1% BSA alone, or the patient serum alone, or finally thecontrol serum (patients not suffering from PNS) and antibodiesconjugated to fluoroescein at the same dilution.

To confirm the immunofluorescence, it is possible to use indirectlabelling by immunoperoxidase. The frozen tissue sections fixed withparaformaldehyde are incubated with 0.3% H₂O₂ (to destroy the intrinsicperoxidase activity) and 10% normal rabbit serum (to avoid thenon-specific binding of the rabbit IgG) or 1% BSA. After incubation for12 h with patient sera diluted to 1/1000 and washing, the sections areincubated for 2 h with biotinylated rabbit anti-human IgG antiserumdiluted to 1/1000 in PBS/1% BSA. The bound human IgGs are visualized byincubation with an avidin-biotin-peroxidase complex (Vectastain ABCcomplex, Vector) and developed with 0.05% DAB (Sigma). The controlsections are obtained with sera of 15 patients without PNS according tothe same protocol.

A—Localization of Proteins of the ULIP Family With the Aid ofAntipeptide Antibodies:

Immunohistochemical labelling was carried out on sections of newborn andadult rat brains. The antipeptide-3 antibody recognizes (an) antigen(s)present in several cell types on sections of newborn and adult ratbrains (FIG. 4). Like the patient anti-CV2 serum, the anti-peptide-4antibodies do not allow the demonstration of any antigen on sections ofnewborn rat brain although they specifically label a subpopulation ofoligodendrocytes in adult rat brain (FIG. 4).

B—Expression of POP-66 in the Course of the Normal Development of theBrain:

FIG. 5 shows that the proliferative nerve cells of the progenitor zonesof the nervous system demonstrated by the accumulation ofbromodeoxyuridine (BrdU) do not express POP-66 although thenon-proliferative cells which correspond to the nerve cells indifferentiation or in migration express it.

EXAMPLE 7 Role of POP-66 in Neuronal Survival

FIG. 6 allows human brain sections of healthy patients and of patientssuffering from PNS to be compared. In the patients suffering from PNSand having circulating anti-CV2 antibodies, a disappearance of theneurons of the dentate gyrus and of pyramidal neurons (central cellband), as well as an intense astrocytic reaction, are observed.

EXAMPLE 8 Characterization of the POP-66 Protein—Identification withULIP-4

Materials and Methods

a) Partial Purification of ULIP-1

Partially purified ULIP-1 was obtained from newborn mouse brains bythree purification steps. These brains were homogenized in 4 volumes ofhomogenization buffer (25 mM sodium phosphate, pH 7.8, 1 mM EGTA, 10μg/ml of leupeptin, 25 μg/ml of aprotinin, and 10 μg/ml of pepstatin.The homogenates were centrifuged for 10 minutes at 400× g. The plugswere resuspended in 2 volumes of homogenization buffer, homogenized andcentrifuged again. The supernatants from two centrifugations werecollected, sonicated and centrifuged for 1 hour at 100,000×g. Thesupernatant (S2) was loaded onto a column of DEAE-Sepharose CL-6B (1.75cm²×26 cm) equilibrated with 100 ml of buffer A (25 mM sodium phosphate,pH 7.8, 1 mM EGTA) at a flow of 30 ml per hour. The proteins were elutedin 300 ml of a 0-250 mM linear gradient of sodium chloride in buffer Aand 5 ml samples were collected. The fractions containing ULIP werecollected and solid ammonium sulphate was added to 20% saturation. Thispool was loaded onto a column of phenyl-Sepharose CL-4B (1.75 cm²×22 cm)which had been previously equilibrated with 100 ml of buffer B (10 mMsodium phosphate, pH 7.8, 1 mM EGTA) containing 20% of saturatedammonium sulphate. The proteins were eluted in a linear gradientdecreasing from 20 to 0% of saturated ammonium sulphate in buffer B. Thefractions containing ULIP were collected and dialysed twice against 20volumes of buffer A. The proteins were concentrated in a small (10 ml)column of DEAE-Sepharose CL-6B and eluted with 400 mM sodium chloride inbuffer A. The eluate was desalted on a Sephadex G-25 (NAP-10) column andconcentrated to a final volume of 0.5 ml by evaporation. In the lastpurification step, the concentrated fraction was chromatographed inthree successive steps, on two Superose 12 FPLC (Fast Protein LiquidChromatography) columns mounted in series, in buffer C (50 mM sodiumphosphate, pH 7.2, 150 mM sodium chloride) at a rate of 0.3 ml/minute.The fractions (0.6 ml) were collected and the fractions enriched in ULIPwere analysed. The presence of ULIP in the successive purification stepswas tested by a one-dimensional Western Blot using an anti-stathminantibody capable of cross-reactivity. The proteins were quantifiedaccording to the method of Bradford.

b) Migration on Electrophoresis Gel:

A one-dimensional electrophoresis was carried out on 13% polyacrylamidegels according to the method of Laemmli. The two-dimensional PAGEelectrophoreses were carried out as described above. Theisoelectro-focusing gels contained 2% of total ampholines, pH 6-8 and3-10 in a ratio of 4:1. The second dimension had been carried out on 10%acrylamide gels. The proteins had been either subjected toimmunoblotting or stained with silver.

c) Western Blot Analysis:

The proteins were transferred from gels to nitrocellulose in buffercontaining 48 mM Tris, 39 mM glycine and 5% of methanol. The membranewas saturated with casein (2.5%) in the immunoblotting solution (12 mMTris-HCl, pH 7.4, 160 mM NaCl, 0.1% Triton X-100) and tested with anantiserum directed against the peptide I of rat stathmin ( 1/10,000dilution) or an antiserum directed against the recombinant ULIP protein(dilution 1/20,000) diluted in an immunoblotting solution containing 1%of casein. The bound antibodies were detected either with a protein Alabelled with ¹²⁵I, and autoradiographed or with anti-rabbit antibodiesbound to peroxidase using the ECL kit (Amersham).

d) Analysis of the Protein Sequence:

The fractions enriched in ULIP were separated on polyacrylamide gels intwo dimensions. The gels are fixed for 30 minutes in 25% ethanol and 10%acetic acid and stained for 3 minutes in 0.1% amido black in 1% aceticacid and 40% methanol. The gels were decolourized in 1% acetic acid andthe spots corresponding to the principal form of ULIP were cut out inthese three gels, collected and digested with 2 mg/ml of Lys Cendoprotease. The peptides eluted from the gel were then separated byHPLC on a DEAE-C18 column with a gradient of 0-55% of acetonitrile in0.1% trifluoro-acetic acid. The peptides were then sequenced accordingto the Edman automatic degradation.

e) Expression In Vitro in a Mammal

1 μg of the Bluescript plasmid containing the entire cDNA coding forULIP-1, ULIP-2, ULIP-3 or ULIP-4 was used to carry out the transcriptionand translation in vitro with the “Reticulocyte lysate” system (Promega)according to the protocol described by the manufacturer. 5 μg of thetotal transcription/translation mixture of 25 μl were analysed onelectrophoresis gel in two dimensions.

Results

Neither the recombinant protein ULIP-1, nor the recombinant proteinsTOAD-64 (ULIP-2) and C-22 (ULIP-3) were recognized by the anti-CV2 sera.In addition, the distribution profile of the spots corresponding toPOP-66 recognized by the anti-CV2 antibodies on electrophoresis in twodimensions does not correspond to the spots recognized by theanti-ULIP-1 antibodies. However, POP-66 is a member of the ULIP familysince the three POP-66 spots are recognized by the anti-pep3 Ab. POP-66therefore corresponds to a member of the family of more basic pH_(i).

After translation in vitro of the four proteins (ULIP-1, 2, 3 and 4), itwas shown that ULIP-4 has the same 2D electrophoretic profile as POP-66and is recognized by the anti-CV2 antibodies (FIG. 7).

For this, the ULIP-4 protein and, as control, the ULIP-2 protein weretranslated in vitro in the presence of ³⁵S methionine from cDNA clonescoding for the entire proteins. The proteins were separated bytwo-dimensional electrophoresis (in the presence of a brain extractproviding the essential references), transferred to nitrocellulose andvisualized:

-   -   by autoradiography to localize the proteins translated in vitro        (translation);    -   by immunoblotting with the CV2 serum.

FIG. 7 shows that the three spots from the in vitro translation ofULIP-4 correspond to the spots recognized by CV2. These spots are notrecognized in the translation of ULIP-2.

The CV2 serum therefore specifically recognizes ULIP-4.

This allowed POP-66 to be identified like ULIP-4.

EXAMPLE 9 Chromosomal Localization of the POP-66/ULIP-4 Protein

Having cloned the cDNA of human ULIP-4, it is then possible to determinethe chromosomal localization of the POP-66/ULIP-4 gene by geneticmapping by in situ isotopic hybridization (Levy and Mattei et al.,1995).

In situ hybridization is carried out on preparations of chromosomesobtained from human lymphocytes stimulated by phytohaemagglutinincultured for 72 hours. 5-Bromodeoxyuridine was added during the last 7hours of culture (60 μg/ml of medium) to ensure a post-hybridizationimage of chromosome bands of good quality. The clone containing aninsert of 1300 base pairs coding for ULIP-4 in the Bluescript vector islabelled with tritium by nick translation with a specific activity of1×10⁸ dpm. μg⁻¹ The radiolabelled probe was hybridized in the metaphasestage at a final concentration of 200 ng per ml of hybridizationsolution. After covering with a Kodak NTB₂ emulsion, the slides wereexposed for 20 days at +4° C. and then developed. To avoid the shiftingof the silver grains during the process, the chromosome blots werepreviously labelled with a Giemsa buffer solution and the metaphaseswere photographed. The visualization of the bands was carried out by the“Giemsa fluorochrome photolysis” (FPG) method and the metaphases wererephotographed before analysis. Of the 100 cells in metaphase examinedafter hybridization in situ, 246 silver grains associated with thechromosomes were counted and 54 amongst these (21.9%) were localized onchromosome 10. The distribution of the grains on this chromosome was notrandom: 39 out of 54 (72.2% of the latter) were localized on theq25.2-q26 region of the long arm of chromosome 10.

The POP-66/ULIP-4 gene is therefore found to be situated on chromosome10 in the q25.2-q26 region. The loci of neurodegenerative diseases andof suppressor genes of tumours involved in different types of cancerhave been localized in this chromosome region. The locus of a braindisease of early origin (infantile onset spinocerebellar ataxia) wasidentified in the 10q24-26 region (Varilo et al., 1996; Nikali et al.,1995). The symptoms of this recessive hereditary degenerative diseasewhich is characterized by ataxia, neuropathy and visual atrophy aresimilar to those observed in patients suffering from paraneoplasticneurological syndromes with circulating anti-CV2 auto-antibodies(Honnorat et al., 1996). On the other hand, 80% of glioblastomas havemutations in this chromosome region and several suppressor loci involvedin different types of tumours (prostate, kidney, small-cell lung cancerand endometrial carcinomas) are localized in this chromosome region.These data support the possibility that POP-66/ULIP-4 plays a crucialrole in neurodegeneration and tumorigenesis.

In this respect, it is notable that the expression of ULIP-1 isregulated upwards in neuroblastoma cells differentiated by retinoic acidand that ULIP-1 and ULIP-3 are regulated upwards but ULIP-4 is regulateddownwards in differentiated PC12 cells in the presence of NGF,suggesting that the stop in cell growth can be linked to expressionlevels of the ULIP proteins.

EXAMPLE 10

Expression of ULIP Proteins in Transfected HeLa Cells

A—Materials and Methods

A flag (EcoRI-ATGGACTACAAGGACGACGATGACAAGG-BamHI) sequence (Kodak) wascloned in the EcoRI site of pSG5 followed by ULIP-1 (EMBEL X87817), basepairs: 309-2023), ULIP-2 (Y10339, base pairs: 23-1741), ULIP-3 (Y09080,base pairs: 269-1991) or ULIP-4 (Y09079, base pairs: 102-1820),respectively. The HeLa cells were cultured in DMEM media (Gibco) towhich 10% of foetal calf serum (v/v) was added. The transfections werecarried out by calcium phosphate precipitation (Maniatis et al., 1978).The HeLa cells were mixed with 5 μg of pSG5flag-ULIP-1, 2, 3 and 4plasmids and 10 μg of pUC18. Twenty-four hours after the transfection,the HeLa cells were fixed with 4% paraformaldehyde and immunolabelledwith different human sera (dilution 1/300), visualized by human anti-IgGantibodies conjugated to FITC (Biosys), or anti-flag antibodies (M2,Kodak) (dilution 1/1000), visualized by anti-rabbit antibodiesconjugated to Texas red (Vector).

Double immunolabelling was carried out on the HeLa cells transfectedwith ULIPs using anti-flag and anti-Pep3 antibodies. In the cellstransfected by any cDNA, 10 to 20% among them showed immunolabellingwith the anti-flag antibodies visualized by the anti-mouse antibodiesconjugated to Texas red.

All the transfected cells were doubly labelled by antibodies directedagainst Pep3 and a peptide common to the four ULIPs is visualized byrabbit anti-IgG antibodies conjugated to fluorescein.

Double immunolabelling was likewise carried out on HeLa cellstransfected with ULIPs using anti-flag and anti-CV2 antibodies. Thehuman sera of patients suffering from PNS with circulating anti-CV2auto-antibodies labelled the cells transfected by ULIP-4, and ananti-CV2 serum likewise labelled the cells transfected by ULIP-3. Nolabelling of the cells transfected by ULIP-4 was detected in the controlsera of patients without cancer or neurological disease.

B) Results

After transfection of HeLa cells with cDNAs labelled by the flags ofULIP-4, 10 to 20% of the cells were strongly reactive with anti-flagantibodies and anti-Pep3 antibodies which recognize the ULIP-4s ofmammals. The transfected cells were not immunolabelled with controlserum of 10 neurological patients without PNS nor with rabbit pre-immuneserum. On the other hand, the cells transfected with small cDNA ofULIP-4 showed an intense immunoreactivity with all the 7 tested sera ofpatients with circulating anti-CV2 auto-antibodies. These sera arenegative on cells transfected with cDNAs of other ULIPs, with theexception of a sera which also recognized the cells transfected withULIP-3 and a serum which also recognized the cells transfected withULIP-1, 3 and 4. No labelling was observed on non-transfected HeLacells, with an anti-CV2 serum.

Table 1 below shows the results of indirect immunofluorescence withdifferent sera on HeLa cells by labelled cDNAs of members of the ULIPfamily. TABLE 1 Serum Neurological Type of No. Symptoms Tumour ULIP-1ULIP-2 ULIP-3 ULIP-4 Anti- — — + + + + Pep3 Pre- — — − − − − immune Pep390-002 PCD, uveitis UC − − + + 93-484 LE Thymoma − − − + 94-590 LE SCLC− − − + 95-700 PEM SCLC + − + + 95-701 PCD Uterine − − − + sarcoma95-706 LE, SCLC − − − + neuropathy 97-040 PCD SCLC − − − + 97-103 PCDSCLC − − − +PCD: paraneoplastic cerebellar degeneration;LE: lymbic encephalitis;PEM: paraneoplastic encephalomyelitis;UC: undifferentiated carcinoma;SCLC: small-cell lung carcinoma.

EXAMPLE 11 Expression of POP-66/ULIP-4 and of Members of the ULIP Familyin Cancers

A—Expression of ULIP-2 and ULIP-3 in Cancers:

1) Materials and Methods: RT-PCR Experiments:

The total RNA was extracted using 1 ml of RNAZOL™B (Bioprobe) accordingto the method of Chomczynski and Sacchi. The quantity of RNA wasdetermined by optical density measured at 260 nm and its purity wasdetermined from the ratio of the absorbances measured at 260 and 280 nm(ratios 1.8-2.0). The integrity of the RNA preparations was additionallychecked by electrophoresis on 1% agarose gel in TBE (0.45 M Tris-borate,10 mM EDTA, pH 8). The specificity of the primers was analyzed bycomparing their sequences with the various gene databanks (EMBL andFASTA). For a relative quantification, the gene coding for G3PDH(glyceraldehyde-3-phosphate dehydrogenase, Clontech), a ubiquitous geneexpressed in numerous tissues including the brain, was co-amplified withthe mRNA tested as an internal standard to check the uniformity of thequantities of RNA in the samples and to test the efficacy of the reversetranscription step for different RNA samples. The primers 5′, 3′ and theoligonucleotides of internal probes of G3PDH were synthesized andpurified by Eurogentec. The total mRNA (1 μg) was denatured (15 minutesat 65° C.) and transcribed in single-stranded cDNA (1 and a half hours,42° C.) in a final volume of 20 μl of buffer (50 mM Tris HCl, 75 mM KCl,pH 8.3, Gibco BRL) containing 5 ng per μl of oligo-dT 12-18 primer(Pharmacia Biotech), 40 units of reverse transcriptase of the Moloneymurine leukaemia virus (Mu-LV) (Gibco BRL), 40 units of RNAsine(Promega), 10 mM DTT (Gibco BRL) and 0.5 mM of each of the triphosphatedeoxynucleotides (Promega). The cDNA samples were diluted to 1/10 indistilled water and the PCR reactions were carried out using 1 μl, 4 μlor 2 μl of cDNA sample for the messenger RNA of ULIP-2 and ULIP-3, in abuffer (50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X100, 0.4% glycerol and800 μM NaCl, pH 9), to which was added 40 μmol of DTT, 3 mmol of MgCl₂,0.2 mmol of each dNTP, 0.4 μM of each selected primer and 2 units ofAmpliTaq DNA polymerase (Promega) in a final volume of 50 μl). Thesamples were then placed in a thermocycler (Biomed-Hybaid), denatured at95° C. for 5 minutes and amplified for 35 cycles (one cycle=95° C.denaturation for 65 seconds, 60° C. hybridization of the primers for 45seconds, 72° C. extension for 4 minutes and 15 minutes final elongationat 72° C. The products were separated by electrophoresis on 1% Seakemagarose gel and the test bands of the RT-PCR products of expected sizeas well as the molecular weight marker scale (100 base pairs) (Promega)were visualized using ethidium bromide staining.

Composition of the oligonucleotide probes used for ULIP-3 PCR 5′ATAGAGGAGCGGATGACG (899) 3′           GCTGTTATGGTCTTCAACTTGTCGG (1092)          GGCCTGTTATGGTCTTCAACTTGTCG (1093)

Composition of the oligonucleotide probes used for ULIP-2 PCR 5′AGGAGGAGTGAAGACCATCG 5227) 3′           CTTATGCCACTCGCTGATGTCC (509).

2) Results

The RT-PCR experiments show that TOAD-64 (ULIP-2) and C-22 (ULIP-3) areexpressed in certain small-cell lung tumours (cf. FIG. 8) and absent inothers, especially in cells of patients who develop paraneoplasticneurological syndromes with a good prognosis.

B—Expression of ULIP-4 in Cancers

1) Materials and Methods

Preparation of the RNA and RT-PCR

The total RNAs are extracted from cerebral tumours preserved in liquidnitrogen according to the conventional RNAZOL™ technique (Bioprobe,France). Reverse transcription was carried out using oligo(dt)₁₈ on 1 μgof total RNA and the PCR was carried out with 1/20 of the volume of themixture for the reverse transcription (RT mix). The primers used forULIP-4 are:

-   5′CATCTGGCTGTCGCTGCAC3′, 5′GCCGCCCCTACCAGAGACC3′, and for GAPDH:    5′GGAGATTCAGTGTGGTGG3′, 5′GGCTCTCCAGAACATCATCC3′. The cDNA was    denatured at 95° C. for five minutes. PCR amplification was carried    out for 30 cycles. ULIP-4: 95° C., 45 sec; 62° C., 45 sec; 72° C.,    45 sec. GAPDH: 95° C., 45 sec; 55° C., 45 sec; 72° C., 45 sec. The    final extension was carried out at 72° C. for 5 minutes.

2) Results

Of the 8 glioblastoma extracts studied, 4 (50%) expressed the messengerRNA of ULIP-4. Conversely, of the 10 oligodendroglioma extracts tested,none expressed the messenger RNA of ULIP-4. This differentialexpression, as a function of the primitive cerebral tumour type, is infavour of a potential role of ULIP-4 in the cell proliferation of thesetumours.

The protein POP-66/ULIP-4 as well as the proteins of the ULIP familycould be expressed in the peripheral tumours (small-cell lung tumour,thymoma, cancer of the breast and of the ovary). Their presence couldtherefore be correlated with a prognosis. The localization of thePOP-66/ULIP-4 gene on the distal part of chromosome 10 confirms this inthe case of cerebral tumours.

Thus, the differential expression of members of the ULIP family intumours such as small-cell lung cancer, although the corresponding ULIPgene is absent in a healthy tissue, as well as the modulation of theexpression of members of the ULIP family obtained during differentiationby the HTLV1 human retrovirus of a medulloblastoma line, suggest theinvolvement of ULIPs in cancerous tumours.

EXAMPLE 12 Production of Specific Antibodies of Each of the Human ULIPProteins

Specific peptides of each member of the ULIP family were synthesized ona multiple peptide synthesis apparatus using F-moc (432A PeptideSynthesizer SYNERGY, Applied Biosystems). The purity was checked bysequence analysis by HPLC and mass spectrometry.

These peptides are:

-   Specific peptide of ULIP-1: G S A R G S P T R P N (11 amino acids)-   Specific peptide of ULIP-2: S S A K T S P A K Q Q A (12 amino acids)-   Specific peptide of ULIP-3: P S A K S S P S K H Q (11 amino acids)-   Specific peptide of ULIP-4: P A R A S C P G K I S (11 amino acids).

1 mg of the synthetic peptide conjugated to limpet haemocyanin, incomplete Freund's adjuvant, was used to immunize rabbits with a boosterdose of 0.5 mg of bound peptide in complete Freund's adjuvant after 4weeks.

The antibodies obtained specifically recognize each member protein ofthe ULIP family.

EXAMPLE 13 Production of Transgenic Animals Expressing ULIP-4

Drosophila fruit flies were transformed by the cDNA of human ULIP-4.

The cDNA of ULIP-4, previously cloned in pbluescript SK-phagemid, wasexcised by Kpn1 and Xba1 enzymatic double digestion. Afterelectrophoresis on agarose gel, the cDNA fragment was purified and thencloned in pUAST, originating from pCaSpeR3, digested by the restrictionenzymes Kpn1 and Xba1. The 10-C plasmid results from the directionalcloning of the cDNA of ULIP-4 in pUAST associated with the mini-whitereporter gene. The 10-C plasmid was injected with a p-delta-2-3 helperplasmid coding for the transposase of the P element active in thegerminal line.

The transformed fruit flies are identified by their red eyes resultingfrom the expression of the mini-white gene. These lines transformed bythe cDNA of ULIP-4 under the control of UASGAL4 regulatory sequencesallow a targeted expression of the cDNA of ULIP-4.

This production of transformed fruit flies allows the role of ULIP-4 tobe studied specifically in different cells and its involvement in humanpathologies to be understood.

BIBLIOGRAPHY

-   Anderson et al., CRC Crit. Rev. Neurobiol., 1987, vol. 3, pp 245-99-   Antoine J. C. et al., Journal of the Neurological Sciences, 1993,    vol. 117, pp 215-223-   Byk et al., Journal of Neuroscience, 1996, vol. 16(2), pp 688-701-   Chomczynksky and Sacchi, Anal. Biochem., 1987, 162: 156-159-   Dalmau et al., Neurology, 1991, vol. 41, pp 1757-64-   Duchemin et al., Dev Neurosci, 1987, vol. 9, pp 61-67-   Graus et al., Neurology, 1985, vol. 35, pp 538-543-   Graus et al., Neurology, 1990, vol. 40, pp 219-22-   Greenlee et al., Ann. Neurol., 1983, vol. 14, pp 609-13-   Hamajima et al., Gene, 1996, vol. 180, pp 157-163-   Hetzel et al., Mayo Clin. Proc., 1990, vol. 65, pp 1558-63-   Honnorat J. et al., Journal of Neurology, Neurosurgery and    Psychiatry, 1996, vol. 61, pp 270-278-   Jaeckle et al., Ann. Neurol., 1985, vol. 18, pp 592-600-   Köhler and Milstein, Nature, 1975, vol. 256, pp 495-497-   Levy N., Mattei M G., 1995, Geneprobs II. A practical approach. B D    Hames and S J Higgins, Oxford University Press, pp 211-243-   Luque et al., Ann. Neurol., 1991, vol. 29, pp 241-51-   Minturn et al., J. Neurosci., 1995, vol. 15, pp 6757-6766-   Nikali et al., Am. J. Hum. Genet., 1995, 56, 1088-1095-   Peltre G., Lapeyre J.; David B., Immunol. Lett., 1982, vol. 5, pp    127-131-   Sambrook et al., Molecular Cloning, a laboratory Manual, 1989,    9.47-9.62-   Varilo et al., Genome Res., 1996, 6:870-875-   Wang L-H et al., J. Neurosci., 1996, vol. 16(9), pp 6197-6207

1. A purified ULIP polypeptide comprising the amino acid sequence of SEQID No.
 8. 2. A composition comprising a purified polypeptide comprisingamino acid sequence SEQ ID No. 8, or a purified fragment thereof,wherein the purified polypeptide or fragment binds to anti-CV2antibodies.
 3. A method for detecting the presence of anti-CV2antibodies in a biological sample, comprising: contacting a biologicalsample with a purified ULIP polypeptide comprising SEQ ID No. 8, or afragment thereof that binds to anti-CV2 antibodies; and detectingspecific immunological complexes optionally formed, the specificimmunological complexes being indicative of the presence of anti-CV2antibodies.
 4. A kit for detection of anti-CV2 antibodies in abiological sample, said kit comprising: at least one purified ULIPpolypeptide comprising SEQ ID No. 8, or a fragment thereof that binds toanti-CV2 antibodies, said polypeptide or fragment optionally attached toa support, and means of visualization of the formation of specificantigen/antibody complexes between an anti-POP-66 auto-antibody and thepurified ULIP polypeptide or fragment and/or means of quantification ofthese complexes.
 5. A method of detecting anti-CV2 antibodies in asubject, said method comprising the steps of: contacting a sample fromthe subject with a purified polypeptide comprising SEQ ID No. 8, or afragment thereof that binds to anti-CV2 antibodies, said contactingcarried out under conditions sufficient to allow the formation ofspecific immunological complexes between the polypeptide or fragmentthereof and anti-CV2 antibodies present within the sample; and detectingthe specific immunological complexes formed; wherein the presence ofimmunological complexes is indicative of the presence of anti-CV2antibodies in said subject.
 6. The method of claim 5, wherein thepolypeptide consists of SEQ ID No.
 8. 7. The method of claim 5,comprising contacting the sample from the subject with a fragment of thepurified polypeptide, said polypeptide consisting of amino acid sequenceSEQ ID No. 8, wherein said fragment binds to anti-CV2 antibodies.
 8. Areagent for identifying anti-CV2 antibodies to a polypeptide in a samplefrom a subject, said reagent comprising a purified peptide comprising afragment of the polypeptide of claim 1 wherein the fragment binds toanti-CV2 antibodies, said fragment attached to a solid support.
 9. A kitfor identifying antibodies in a sample from a subject to a polypeptidecomprising amino acid sequence of SEQ ID No. 8, said kit comprising afragment of said polypeptide that binds to anti-CV2 antibodies.
 10. Thekit of claim 9, wherein the kit further comprises means of visualizingformation of complexes between said fragment and antibodies to thepolypeptide comprising amino acid sequence of SEQ ID No.
 8. 11. The kitof claim 9, wherein the fragment of said polypeptide is purified.
 12. Areagent for identifying anti-CV2 antibodies in a sample from a subject,said reagent comprising: a purified polypeptide comprising amino acidsequence SEQ ID NO:8, said polypeptide attached to a solid support.